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Abstract

Background: Widespread resistance of Neisseria gonorrhoeae to penicillin, tetracycline, and fluoroquinolones has challenged effective treatment and control; recent international case reports of cefixime, ceftriaxone, and azithromycin resistance suggest that the remaining treatment options are now additionally threatened. To explore trends in antimicrobial susceptibility of N. gonorrhoeae, we reviewed provincial laboratory data from British Columbia, 2006 to 2011.

Methods: Susceptibility testing was performed for all N. gonorrhoeae isolates detected in-house or forwarded to the reference laboratory. Resistance or intermediate resistance (nonsusceptibility) was defined by standard breakpoints for penicillin, tetracycline, ciprofloxacin, and spectinomycin. Elevated minimum inhibitory concentrations (MICs) at serial dilutions of 0.064 μg/mL or greater were explored for cefixime/ceftriaxone and 0.5 μg/mL or greater for azithromycin. Nonsusceptibility/elevated MIC was compared by year, site of infection, sex, and age.

Results: A total of 1837 isolates representing 22% of all reported gonorrhea cases were analyzed. Nonsusceptibility to penicillin was established at baseline. Nonsusceptibility to tetracycline and ciprofloxacin increased over the study period, reaching 96% and 36%, respectively, in 2011. Sixteen isolates (1%) had a cefixime MIC of 0.25 μg/mL (none ≥0.5), none had a ceftriaxone MIC of 0.25 μg/mL or greater, and 15 (1%) had an azithromycin MIC of 2.0 μg/mL or greater. Elevated MIC of these agents showed an increasing trend over time. Nonsusceptibility and elevated MIC were consistently highest at the rectal and pharyngeal sites and higher in isolates from males, including when stratified to the pharyngeal site.

Interpretation: Increases in elevated MIC of cefixime/ceftriaxone/azithromycin were superimposed on a background of established resistance to penicillin, tetracycline, and ciprofloxacin and may signal impending gonococcal resistance to first-line treatments. Ongoing surveillance will inform timely shifts in treatment recommendations.

Antimicrobial resistance (AMR) of Neisseria gonorrhoeae has continually challenged effective treatment and control since the introduction of penicillin in the mid-20th century.1 Penicillin and tetracycline resistance were first identified during the 1970s and have subsequently become ubiquitous.1,2 More recently, resistance to fluoroquinolones emerged, further limiting treatment options.1 Current treatment guidelines for N. gonorrhoeae in Canada, as elsewhere, therefore recommend third-generation cephalosporins as first-line treatments, with cotreatment using a drug from another class.3,4

Since 2000, however, there have been multiple case reports of reduced susceptibility and treatment failure with cefixime, suggesting that the effectiveness of cephalosporins in treating gonorrhea is now additionally threatened.5,6 Recent international data reveal a rise in the percentage of isolates demonstrating elevated minimum inhibitory concentration (MIC) of cephalosporins (defined as ≥0.25 μg/mL for cefixime, ≥0.125 μg/mL for ceftriaxone): England and Wales (0% cefixime/ceftriaxone in 2004 vs. 1% cefixime and 0.5% ceftriaxone in 2008),7 the United States (0.2% cefixime and 0.1% ceftriaxone in 2000, 1% cefixime and 0.3% ceftriaxone in 2010, 2% cefixime and 0.5% ceftriaxone in 2011),8,9 and Canada (0.2% cefixime/ceftriaxone in 2001 vs. 1% cefixime and 3% ceftriaxone in 2009).10,11 In the United States, decreased susceptibility to cephalosporins has been observed almost exclusively among samples from men who have sex with men (MSM).8,9 In 2009, the first high-level ceftriaxone-resistant (MIC 2 μg/mL) and cefixime-resistant (MIC 8 μg/mL) gonococcal isolate was identified in Japan, from a pharyngeal specimen.12 A subsequent high-level cefixime (MIC 4 μg/mL) and ceftriaxone-resistant (MIC 2 μg/mL) strain was identified in France in 2010, in a urethral specimen from a man who was sexually active with men.13 Although these Japanese and French isolates retained susceptibility to azithromycin (MIC <2 μg/mL),12,13 other recent cases have demonstrated high-level resistance to this agent (≥8 μg/mL).14–18 In European surveillance networks, 2% to 13% of isolates had a MIC of 1 μg/mL or greater to azithromycin in 2006 to 2009.19,20

In light of these concerning signals of diminishing treatment options for N. gonorrhoeae, there is an ongoing need for systematic surveillance of gonococcal resistance.1,9,21 Routine AMR testing of N. gonorrhoeae isolates has been in place since 1991 at the British Columbia (BC) Provincial Public Health Microbiology and Reference Laboratory (PHMRL) and has included cephalosporin and azithromycin sensitivity testing since 2006. The western Pacific region is of particular interest for monitoring the emergence of N. gonorrhoeae AMR because of its proximity to Asia; western North America served as a harbinger of broad fluoroquinolone resistance in Canada and the United States,1 and data from the United States forecast a similar pattern for cephalosporins.8,9 Here we illustrate recent 6-year trends in reduced susceptibility to cephalosporins and azithromycin among N. gonorrhoeae isolates in BC.

MATERIALS AND METHODS

The BC PHMRL performs approximately 15% to 20% of all gonorrhea tests in the province, receiving specimens predominantly from Provincial Sexually Transmitted Infection Clinic sites at the BC Centre for Disease Control; regional public health, youth, reproductive, and sexual health clinics; and hospitals throughout the province. At the PHMRL, gonorrhea may be detected by nucleic acid amplification testing (NAAT) or conventional culture diagnostic methods. Culture testing is preferentially used for rectal and pharyngeal specimens and for all specimens from contacts to gonorrhea as well as patients who are symptomatic, not responding to treatment, or presenting for treatment after an initial NAAT-positive test result. Antimicrobial resistance testing is routinely performed for all N. gonorrhoeae isolated by culture from clinical specimens. The PHMRL additionally receives gonorrhea isolates forwarded for susceptibility testing from community- or hospital-based laboratories in BC. For present analyses, laboratory data for all tested isolates from March 9, 2006, to December 31, 2011, were extracted.

Analysis

Data were analyzed by isolate. Patient age and sex, as well as site of infection, were compared between AMR-tested isolates and reported gonorrhea cases for the same period, using χ2 test. Clinical and Laboratory Standards Institute (CLSI) guidelines were used to define MIC breakpoints for resistant and intermediate categories: penicillin (≥2 μg/mL, 0.125–1 μg/mL), tetracycline (≥2 μg/mL, 0.5–1 μg/mL), ciprofloxacin (≥1 μg/mL, 0.125–0.5 μg/mL), and spectinomycin (≥128 μg/mL, 64 μg/mL).22 Resistant and intermediate categories were collapsed for some analyses, hereafter termed nonsusceptibility. The CLSI defines an MIC of 0.25 μg/mL or less as susceptible to cefixime and ceftriaxone but does not clearly define intermediate/resistant categories because of lack of data.22 No standardized CLSI criteria exist for azithromycin; however, some sources propose an MIC of 2 μg/mL or greater as the threshold for nonsusceptibility.22,23 Minimum inhibitory concentrations were thus explored at 2 to 3 serial dilutions below each of these limits: “elevated MIC” was defined as 0.064 μg/mL or greater for cefixime/ceftriaxone and 0.5 μg/mL or greater for azithromycin. In consideration of distinct treatment recommendations for pharyngeal versus anogenital gonorrhea (in Canada, 250 mg intramuscular single-dose ceftriaxone is the preferred treatment as of December 2011 and emphasized particularly for MSM and for pharyngeal infections in all patients irrespective of sex/sexual orientation3) and to account for behavioral risk factors, results were stratified by site of infection: urethra, cervix, rectum, and throat. Nonsusceptibility and elevated MIC were examined with particular focus on temporal trends, compared across sex and age categories, and tested for significance by χ2 test or Fisher exact test, where appropriate (2 sided; P < 0.05 considered statistically significant). We also investigated whether isolates exhibiting elevated MIC of cefixime, ceftriaxone, or azithromycin had reduced susceptibility to other antimicrobials currently used for gonorrhea treatment.

RESULTS

From 2006 to 2011, there were 8353 reported cases of gonorrhea in BC. A total of 1837 isolates were available for analysis; thus, AMR-tested isolates represent approximately 22% of all reported cases in the province. The number of AMR-tested isolates steadily increased by year, from 193 in 2006 to 501 in 2011. During this period, patients providing AMR-tested isolates were slightly older than reported gonorrhea cases (57% vs. 47% were ≥30 years of age; P < 0.001) and included more men (85% vs. 66%; P < 0.001). Rectal and pharyngeal gonorrhea were also overrepresented in AMR-tested isolates (among AMR-tested isolates, 51% were from the urethra, 13% cervix, 23% rectum, and 12% pharynx vs. 62%, 26%, 7%, and 5%, respectively, among all provincial reported cases; P < 0.001). Approximately 2% of all isolates were from other sites and excluded from further analysis. Ninety-nine percent of rectal isolates and 91% of pharyngeal isolates were from men. Men providing AMR-tested isolates were older than women (60% vs. 38% were ≥30 years of age; P < 0.001). Sixty-nine percent of AMR-tested isolates came from the Provincial Sexually Transmitted Infection Clinic sites; this proportion fluctuated slightly from year to year but showed no apparent trend.

All isolates were susceptible to spectinomycin. Susceptibility patterns for all other antimicrobial agents are presented in Table 1. Nonsusceptibility (defined as intermediate resistance or resistance) to penicillin and tetracycline was established at baseline and gradually increased over the period of study, with more than 95% of isolates nonsusceptible to both drugs in 2011 (χ2 tests for trend: P = 0.002 and P < 0.001, respectively). Ciprofloxacin nonsusceptibility increased from 25% in 2006 to 69% in 2010 and dropped to 36% in 2011, but maintained a general increasing trend over the total study period (P < 0.001). Nonsusceptibility to penicillin and tetracycline was higher among males than among females (P < 0.001 for both) and increased with age (P < 0.001 for both), whereas sex-related (P = 0.71) and age-related (P = 0.12) patterns were not apparent for ciprofloxacin. Age-related trends for penicillin and tetracycline persisted after restricting to males (data not shown; P = 0.07 for penicillin and P = 0.02 for tetracycline). Nonsusceptibility to all 3 antimicrobials was highest at the rectal site, next highest at urethral and pharyngeal sites, and lowest at the cervical site; however, this gradient was less pronounced for ciprofloxacin (P = 0.93) than for penicillin (P < 0.001) and tetracycline (P < 0.001). Further stratification of nonsusceptibility at the pharyngeal site by sex showed a consistent disparity, although differences were not statistically significant (P = 0.46 for penicillin, P = 0.26 for tetracycline, P = 0.21 for ciprofloxacin); the small sample of rectal specimens from females (n = 5) precluded further stratification of those results by sex.

Three hundred sixty-seven isolates (20%) had elevated cefixime MIC: 113 (6%) had an MIC of 0.064 μg/mL, 238 (13%) had an MIC of 0.125 μg/mL, and 16 (1%) had an MIC of 0.25 μg/mL; none had an MIC of 0.5 μg/mL or greater. Two hundred fifty-four (14%) isolates had elevated ceftriaxone MIC: 227 (13%) had an MIC of 0.064 μg/mL and 27 (1%) had an MIC of 0.125 μg/mL; none had an MIC of 0.25 μg/mL or greater. Nine hundred seventy-one isolates (54%) had elevated azithromycin MIC: 649 (36%) had an MIC of 0.5 μg/mL, 307 (17%) had an MIC of 1.0 μg/mL, and 15 (1%) had an MIC of 2.0 μg/mL or greater (5 with an MIC of 2.0 μg/mL, 1 with 4.0, 5 with 8.0, and 4 with 16.0). Trends in elevated MIC of cefixime, ceftriaxone, and azithromycin are shown in Figures 1, 2, and 3, respectively. A temporal increase in elevated MIC was evident for all 3 of these agents (χ2 tests for trend: P < 0.001). The percentage of isolates with elevated MIC generally increased over time across all 4 sites of infection and for both cephalosporins and azithromycin, with the exception of elevated MIC of cephalosporins in 2011, which decreased among urethral, rectal, and pharyngeal isolates but not cervical isolates. A temporal increase was also observed for the highest elevated MIC of cefixime (0.25 μg/mL): 1% (1/193 isolates) in 2006, 0% in 2007 (0/230), 1% in 2008 (2/244), 1% in 2009 (3/302), 1% in 2010 (2/337), and 2% in 2011 (8/501). Trends in elevated MIC were consistent between isolates coming from Provincial Sexually Transmitted Infection Clinics and those referred into the laboratory from other clinics (data not shown).

Elevated MICs were consistently highest at the rectal site, next highest at the pharyngeal site, lower at the urethral site, and lowest at the cervical site (P < 0.001 for all 3 tests) (Figs. 1, 2, and 3; Table 1). Analysis of elevated MIC by sex showed similar disparities; over the course of the entire analysis period, 21% of isolates from males versus 15% from females had a cefixime MIC of 0.064 μg/mL or greater (P = 0.002; 26% among males vs. 16% among females at the pharyngeal site, P = 0.42), 15% versus 8% had a ceftriaxone MIC of 0.064 μg/mL or greater (P = 0.001; 16% vs. 5% pharyngeal, P = 0.32), and 58% versus 31% had an azithromycin MIC of 0.5 μg/mL or greater (P < 0.001; 62% vs. 42% pharyngeal, P = 0.09). Of the 16 isolates with a cefixime MIC of 0.25 μg/mL, 15 were from males (6 urethral, 6 rectal, and 3 pharyngeal specimens) and 1 was from a female (cervical specimen). Elevated MIC of azithromycin showed an increasing trend with age (P < 0.001); this trend persisted but was diminished after restricting to males (data not shown; P = 0.24). Age-related trends were not apparent for cefixime (P = 0.91) or ceftriaxone (P = 0.48).

Patterns of multidrug susceptibility are shown in Table 2. Among isolates with elevated cefixime MIC, 68% also exhibited elevated ceftriaxone MIC, 91% exhibited elevated azithromycin MIC, and 99% exhibited nonsusceptibility to ciprofloxacin. Similar patterns were apparent for isolates with elevated ceftriaxone MIC. By contrast, only 54% of isolates with elevated azithromycin MIC were nonsusceptible to ciprofloxacin.

DISCUSSION

This report on recent surveillance data from the Canadian province of BC demonstrates a temporal rise in elevated MIC of cefixime, ceftriaxone, and azithromycin between 2006 and 2011. A consistent disparity in reduced susceptibility by site of infection (and sex) was apparent, whereby the percentage of isolates with elevated MIC was highest in rectal specimens, next highest in pharyngeal specimens, lower in urethral specimens, and lowest in cervical specimens. It should be noted that the clinical significance of elevated MIC in the absence of established thresholds for resistance remains unclear. Nonetheless, stepwise exploration of sequential MIC values can be informative, as shown here. The upward MIC “creep” in relation to these first-line treatments was superimposed on a background of established resistance to penicillin, tetracycline, and ciprofloxacin and could be a strong warning sign of impending resistance to these agents.

Evidence of MIC creep to azithromycin found in our analysis is consistent with recent reports of emerging azithromycin resistance in Scotland,17 England, and Wales.18 These findings contrast, however, with other reports from Argentina,16 the United States,14,23 and Europe,19 which suggest limited or sporadic resistance, with no consistent trend. Elevated MIC of azithromycin may be emerging due to low-level serum concentration from the treatment of chlamydia (i.e., with 1-g dosage) or its use for respiratory tract infections in the community.1,17 Rates of community use of azithromycin increased in BC from 1996 to 2005 (∼0.1–0.5 daily defined doses [DDDs] per 1000 population/d) and thereafter remained steady (∼0.5 DDD/1000 population/d).24 With its long elimination half-life, azithromycin has demonstrated a greater propensity to select for resistant bacteria compared with other classes of antibiotics, and hence, its widespread use has been discouraged.25

Minimum inhibitory concentration creep in relation to cephalosporins was first reported in Asia in the early 2000s and subsequently observed in Australia, Europe, and, most recently, North America.6–11,21 The exact mechanism of cephalosporin resistance remains uncertain. Most N. gonorrhoeae strains with elevated MIC show multiple chromosomal alterations, including a change at penicillin-binding protein 2—notably also associated with penicillin resistance26—although this mutation seems to be neither necessary nor sufficient to confer high-level resistance, and it likely affects oral cephalosporins (i.e., cefixime) more than injectable ceftriaxone.1,21 Use of third-generation cephalosporins in community settings in BC remains low and steady (below 0.1 DDD/1000 population/d, with little change in trend from 1996 to 2008),27 suggesting that cephalosporin MIC creep observed in our analysis may be attributed to importation from other regions. Elevated MIC values used in our analysis are lower than those currently used as alert values in the United States. Although the significance of MIC dilutions 2- and 4-fold below the threshold of susceptibility is uncertain, the consistent trends we revealed at these low levels suggest that exploration of such values may be worthwhile in other geographic settings.

Our finding of higher rates of elevated cephalosporin and azithromycin MIC at rectal and pharyngeal sites may represent transmission within sexual networks of gay men and other MSM, who first experienced prevalent fluoroquinolone resistance in North America and who demonstrated higher rates of elevated MIC of cephalosporins in recent data from the United States.8,9 Although we were unable to assess trends by sexual behaviors, most of the pharyngeal and rectal isolates in our data may be assumed to be from MSM (91% and 99% of these isolates, respectively, were from males). Notably, the disparity between nonsusceptibility or elevated MIC at rectal/pharyngeal versus urethral sites in our analysis was most pronounced for cephalosporins, and azithromycin, antibiotics for which patterns of resistance are still emerging. Together, these results substantiate a strategy of distinct treatment recommendations for MSM; higher antibiotic doses or cotreatment with an antibiotic from a different class may help avert further emergence of resistance. Another strategy would entail more systematic diagnosis and treatment of pharyngeal gonorrhea, which is predominantly asymptomatic.28

Testing guidelines in BC result in the preferential use of culture for diagnosis of gonorrhea in patients who have known risk factors or are symptomatic. Thus, as with other similar AMR surveillance systems, our sample cannot be considered representative of the total population of gonorrhea cases, as illustrated by significant differences in age, sex, and site of infection. This would likely have the effect of overestimating rates of nonsusceptibility—assuming cases of treatment failure and reinfection are oversampled—however, this selection bias is not expected to change with time; therefore, these data remain valuable for trend estimation. As acknowledged earlier, because our analyses are based on laboratory data, we do not have accompanying behavioral or sociodemographic information. This limitation may be addressed, in part, through the routine linkage of detailed epidemiologic information, clinical details, and treatment outcomes, with laboratory results. Although passive in nature, most isolates in the BC surveillance system come from one provincial sexually transmitted infection clinic in Vancouver, and this further limits the geographic generalizability of these results.

Our results add to the growing literature that demonstrates decreasing susceptibility to cephalosporins globally. The anticipated emergence of cephalosporin resistance places high demand on effective prevention and control measures, and in light of diminishing treatment options, prioritization of these measures warrants careful consideration. Access to systematic testing should be bolstered, particularly among MSM and inclusive of asymptomatic clients with known risk factors for sexually transmitted infections. Tests of cure (preferably with culture and sensitivity methods) should furthermore be considered for all clinical treatment failures and in sentinel surveillance programs. Finally, given an increased reliance upon NAAT for gonorrhea detection, novel methods for detecting genotypic resistance in NAAT-positive specimens would greatly improve current surveillance and drug resistance control efforts. Although only sporadic gonorrhea cases have shown failure to respond to cephalosporins and azithromycin to date, these may be underreported; the MIC creep observed in these surveillance data supports that the emergence of resistance to these drugs can be expected in the near future. In this context, ongoing and strengthened gonorrhea resistance surveillance offers the opportunity to adjust treatment approaches before widespread cephalosporin and azithromycin treatment failures occur.

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